U.S. patent application number 11/235557 was filed with the patent office on 2006-07-13 for novel members of the steroid/thyroid superfamily and uses therefor.
This patent application is currently assigned to The Salk Institute for Biological Studies. Invention is credited to Bruce Blumberg, Ronald M. Evans, Barry M. Forman, Steven A. Kliewer, Estelita S. Ong.
Application Number | 20060154335 11/235557 |
Document ID | / |
Family ID | 23032190 |
Filed Date | 2006-07-13 |
United States Patent
Application |
20060154335 |
Kind Code |
A1 |
Evans; Ronald M. ; et
al. |
July 13, 2006 |
Novel members of the steroid/thyroid superfamily and uses
therefor
Abstract
Novel peroxisome proliferators-activated receptor subunits
designated PPAR.gamma. and PPAR.delta. are described. Nucleic acid
sequences encoding the receptor subunits, expression vectors
containing such sequences and host cells transformed with such
vectors are also disclosed, as are heterodimeric PPAR receptors
comprising at least one of the invention subunits, and methods for
the expression of such novel receptors, and various uses
therefor.
Inventors: |
Evans; Ronald M.; (La Jolla,
CA) ; Forman; Barry M.; (Irvine, CA) ;
Kliewer; Steven A.; (Cary, NC) ; Ong; Estelita
S.; (San Diego, CA) ; Blumberg; Bruce;
(Irvine, CA) |
Correspondence
Address: |
FOLEY & LARDNER LLP
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
The Salk Institute for Biological
Studies
|
Family ID: |
23032190 |
Appl. No.: |
11/235557 |
Filed: |
September 26, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
08484215 |
Jun 7, 1995 |
|
|
|
11235557 |
Sep 26, 2005 |
|
|
|
08270643 |
Jul 1, 1994 |
|
|
|
08484215 |
Jun 7, 1995 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/705 20130101;
C07K 14/70567 20130101; G01N 2500/00 20130101; G01N 33/74
20130101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C07K 14/705 20060101
C07K014/705; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06 |
Claims
1. An isolated mammalian peroxisome proliferator-activated receptor
subunit protein of the gamma subtype (PPAR-.gamma.), or functional
fragments thereof.
2. A protein according to claim 1 wherein said protein is
characterized by: the ability to repress PPAR.alpha.-mediated
responses activated by Wy 14,643; being activated by LY-171883, but
not linoleic acid; and being encoded by nucleic acid which
hybridizes under high stringency conditions to a nucleic acid which
encodes the amino acid sequence set forth in SEQ ID NO:2.
3. A protein according to claim I wherein said PPAR-.gamma. has
more than 95% amino acid identity with SEQ ID NO: 2.
4. A protein according to claim 1 having an amino acid sequence
substantially the same as set forth in SEQ ID NO:2.
5. A protein according to claim 1 having the same amino acid
sequence as set forth in SEQ ID NO:2.
6. An isolated mammalian peroxisome proliferator-activated receptor
subunit protein of the delta subtype (PPAR-.delta.), or functional
fragments thereof.
7. A protein according to claim 6 wherein said protein is
characterized by: the ability to repress PPAR.alpha.-mediated
responses activated by Wy 14,643; being activated by LY-171883, but
not linoleic acid; and being encoded by nucleic acid which
hybridizes under high stringency conditions to a nucleic acid which
encodes the amino acid sequence set forth in SEQ ID NO:4.
8. A protein according to claim 6 wherein said PPAR-.delta. has
more than 95% amino acid identity with SEQ ID NO: 4.
9. A protein according to claim 6 having an amino acid sequence
substantially the same as set forth in SEQ ID NO:4.
10. A protein according to claim 6 having the same amino acid
sequence as set forth in SEQ ID NO:4.
11. A heterodimer complex comprising a receptor according to claim
1 and an isoform of RXR.
12. A heterodimer complex according to claim 11 wherein said
isoform of RXR is selected from the group consisting of RXR.alpha.,
RXR.beta. and RXR.gamma..
13. A heterodimer complex comprising a receptor according to claim
6 and an isoform of RXR.
14. A heterodimer complex according to claim 13 wherein said
isoform of RXR is selected from the group consisting of RXR.alpha.,
RXR.beta. and RXR.gamma..
15. An antibody generated against the receptor of claim 1.
16. An antibody generated against the receptor of claim 6.
17. A method for identifying compounds potentially useful for the
treatment of diseases modulated by a PPAR-.gamma. according to
claim 1, said method comprising determining whether a compound
interacts directly with said PPAR-.gamma., thereby identifying
compounds that interact directly with PPAR-.gamma. as useful for
the treatment of diseases modulated by PPAR-.gamma..
18. A method for identifying compounds potentially useful for the
treatment of diseases modulated by a PPAR-.gamma. according to
claim 1, said method comprising determining whether a compound
activates said PPAR-.gamma., thereby identifying compounds that
activate PPAR-.gamma. as useful for the treatment of diseases
modulated by PPAR-.gamma..
19. A method for identifying compounds potentially useful for the
treatment of diseases modulated by a PPAR-.delta. according to
claim 6, said method comprising determining whether a compound
interacts directly with said PPAR-.delta., thereby identifying
compounds that interact directly with PPAR-.delta. as useful for
the treatment of diseases modulated by PPAR-.delta..
20. A method for identifying compounds potentially useful for the
treatment of diseases modulated by a PPAR-.delta. according to
claim 6, said method comprising determining whether a compound
activates said PPAR-.delta., thereby identifying compounds that
activate PPAR-.delta. as useful for the treatment of diseases
modulated by PPAR-.delta..
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 08/484,215, filed Jun. 7, 1995, now pending, which is a
continuation-in-part of U.S. application Ser. No. 08/270,643, filed
Jul. 1, 1994, now abandoned, the entire contents of each of which
are hereby incorporated by reference herein.
FIELD OF INVENTION
[0002] The present invention relates to novel members of the
steroid/thyroid superfamily of receptors, as well as uses
therefor.
BACKGROUND OF THE INVENTION
[0003] Peroxisome proliferators are a structurally diverse group of
compounds which, when administered to rodents, elicit dramatic
increases in the size and number of hepatic and renal peroxisomes,
as well as concomitant increases in the capacity of peroxisomes to
metabolize fatty acids via increased expression of the enzymes
required for the .beta.-oxidation cycle (Lazarow and Fujiki, Ann.
Rev. Cell Biol. 1:489-530 (1985); Vamecq and Draye, Essays Biochem.
24:1115-225 (1989); and Nelali et al., Cancer Res. 48:5316-5324
(1988)). Chemicals included in this group are the fibrate class of
hypolipidermic drugs, herbicides, and phthalate plasticizers (Reddy
and Lalwani, Crit. Rev. Toxicol. 12:1-58 (1983)). Peroxisome
proliferation can also be elicited by dietary or physiological
factors such as a high-fat diet and cold acclimatization.
[0004] Insight into the mechanism whereby peroxisome proliferators
exert their pleiotropic effects was provided by the identification
of a member of the nuclear hormone receptor superfamily activated
by these chemicals (Isseman and Green, Nature 347-645-650 (1990)).
This receptor, termed peroxisome proliferator activated receptor
alpha (PPAR.alpha.), was subsequently shown to be activated by a
variety of medium and long-chain fatty acids and to stimulate
expression of the genes encoding rat acyl-CoA oxidase and
hydratase-dehydrogenase (enzymes required for peroxisomal
.beta.-oxidation), as well as rabbit cytochrome P450 4A6, a fatty
acid .omega.-hydroxylase (Gottlicher et al., Proc. Natl. Acad. Sci.
USA 89:4653-4657 (1992); Tugwood et al., EMBO J. 11:433-439 (1992);
Bardot et al., Biochem. Biophys. Res. Comm. 192:37-45 (1993);
Muerhoff et al., J. Biol. Chem. 267:19051-19053 (1992); and Marcus
et al., Proc. Natl. Acad. Sci. USA 90(12):5723-5727 (1993). The
foregoing references support a physiological role for PPAR.alpha.
in the regulation of lipid metabolism. PPAR.alpha. activates
transcription by binding to DNA sequence elements, termed
peroxisome proliferator response elements (PPRE), as a heterodimer
with the retinoid X receptor. The retinoid X receptor is activated
by 9-cis retinoic acid (see Kliewer et al., Nature 358:771-774
(1992), Gearing et al., Proc. Natl. Acad. Sci. USA 90:1440-1444
(1993), Keller et al., Proc. Natl. Acad. Sci. USA 90:2160-2164
(1993), Heyman et al., Cell 68:397-406 (1992), and Levin et al.,
Nature 355:359-361 (1992)). Since the PPAR.alpha.-RXR complex can
be activated by peroxisome proliferators and/or 9-cis retinoic
acid, the retinoid and fatty acid signaling pathways are seen to
converge in modulating lipid metabolism.
BRIEF DESCRIPTION OF THE INVENTION
[0005] In accordance with the present invention, there are provided
isolated mammalian peroxisome proliferators-activated receptor
subunit proteins of the .gamma. and .alpha. subtypes, and
functional fragments thereof. In addition, there are provided
isolated nucleic acids encoding mammalian peroxisome
proliferator-activated receptor subunit proteins, as well as
fragments thereof. There are also provided vectors containing the
above-described nucleic acids, as well as cells containing such
nucleic acids and/or vectors.
[0006] The present invention also provides methods for the
recombinant production of mammalian peroxisome
proliferator-activated receptor proteins comprising at least one
PPAR subunit protein of the .gamma. and .delta. subtype, and
functional fragments thereof, as well as methods to identify clones
encoding the above-described receptor subunit proteins, and
functional fragments thereof.
[0007] Also provided by the present invention are methods for
screening compounds to determine those which bind to mammalian
peroxisome proliferator-activated receptor proteins comprising at
least one PPAR subunit protein of the .gamma. or .delta. subtype,
or functional fragments thereof, as well as bioassays for
evaluating whether test compounds are agonists or antagonists for
receptor proteins of the invention, or functional modified forms of
said receptor protein(s).
BRIEF DESCRIPTION OF THE FIGS.
[0008] FIG. 1 presents a schematic comparison of the members of the
PPAR gene family using mPPAR.delta. as a reference. Comparisons
among the different domains of the proteins are expressed as
percent amino acid identity.
[0009] FIG. 2 demonstrates that PPAR.gamma. and PPAR.delta. fail to
respond to the peroxisome proliferator Wy 14,643. CV-1 cells were
cotransfected with reporter plasmid PPRE.sub.3-TK-LUC and either no
receptor expression plasmid (-), CMX-PPAR.alpha., CMX-PPAR.gamma.,
or CMX-PPAR.delta. and then incubated in either the absence (-) or
presence (+) of 5 .mu.M Wy 14,643. Luciferase activities are
expressed as percentages of the maximal response where 100% is the
activity obtained with PPAR.alpha. in the presence of 5 .mu.M Wy
14,643.
[0010] FIG. 3 illustrates the ability of PPAR.gamma. and
PPAR.delta. to repress PPAR.alpha.-mediated responsiveness to Wy
14,643. CV-1 cells were cotransfected with reporter plasmid
PPRE.sub.3-TK-LUC and either no receptor expression plasmid (NONE)
or CMX-PPAR.alpha. (10 ng) in either the absence or presence of
CMX-PPAR.gamma. (100 ng) or CMX-PPAR.delta. (100 ng). Cells were
then incubated in either the absence (-) or presence (+) of 5 .mu.M
Wy 14,643. Luciferase activities are presented as fold-activation
relative to cells which were not transfected with receptor
expression plasmid and were not treated with Wy 14,643.
[0011] FIG. 4 demonstrates that PPAR isoforms are pharmacologically
distinct. CV-1 cells were cotransfected with reporter plasmid
PPRE.sub.3-TK-LUC and either no receptor expression plasmid (-),
CMX-PPAR.alpha., CMX-PPAR.gamma., or CMX-PPAR.delta. in either the
absence or presence of 5 .mu.M Wy 14,643 (WY), 3O .mu.M linoleic
acid (C18:2), or 30 .mu.M LY-171883 (LY). Luciferase activities are
presented as the fold activation achieved in compound-treated
versus mock-treated cells. Similar results were obtained in
triplicate in three independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0012] Two novel PPAR receptor subunits have been cloned and
characterized. These novel .gamma. and .delta. isoforms (subunits)
together with the a subunit display marked differences in their
responsiveness to peroxisome proliferators and fatty acids, as well
as differences in their temporal and spatial patterns of
expression. These observations suggest a broad role for the PPAR
family during development and in adult physiology.
[0013] The existence of multiple PPAR isoforms with distinct
expression patterns has been found to correlate with the fact that
the three isoforms have different ligand specificities. Indeed, the
PPAR isoforms are shown herein to be pharmacologically distinct.
Thus, PPAR.alpha., PPAR.gamma. and PPAR.delta. are most efficiently
activated by Wy 14,643, LY-171883, and linoleic acid, respectively.
Remarkably, Wy 14,643, which results in approximately 100-fold
induction in reporter expression in the presence of PPAR.alpha.,
fails to activate either PPAR.gamma. or PPAR.delta..
[0014] With regard to this differential responsiveness to
activators of peroxisome proliferation, the relationship among the
PPAR isoforms may be analogous to that between the glucocorticoid
and mineralocorticoid receptors (GR and MR, respectively). While
both receptors can bind to the same response element, and both
respond to mineralocorticoids and corticosteroids, MR and GR
display differential sensitivities to aldosterone and specific
glucocorticoids such as dexamethasone, respectively (Arriza et al.,
Neuron 1:887-900 (1988)). Thus, the ratio of these receptors to
their ligands provides a means of determining tissue-specific
expression of target genes. Similarly, the existence of multiple
PPAR isoforms with overlapping ligand specificities may provide the
means for tissue-specific regulation of gene expression by
peroxisome proliferators and fatty acids.
[0015] In addition to their differential responsiveness to
peroxisome proliferators, the three PPAR isoforms also display
distinct yet overlapping expression patterns. As previously shown,
PPAR.alpha. mRNA is abundant in liver and kidney (Isseman and
Green, supra Beck et al., Proc. R. Soc. Lond. 247:83-87 (1992)),
tissues in which peroxisome proliferators result in dramatic
increases in the numbers of peroxisomes and concomitant increases
in peroxisomal .beta.-oxidation (Nemali et al., supra). In
contrast, the levels of PPAR.gamma. mRNA and PPAR.delta. mRNA,
which can act as dominant repressors of PPAR.alpha.-mediated
responsiveness to Wy 14,643, are bow in these tissues. Thus, a
pattern emerges in which tissues that are most responsive to
peroxisome proliferators such as Wy 14,643 are observed to express
high amounts of PPAR.alpha. mRNA and relatively low amounts of
PPAR.gamma. mRNA and PPAR.delta. mRNA. These data suggest that the
ratio of the PPAR isoforms is likely to play a critical role in
establishing the degree of responsiveness of tissues to specific
peroxisome proliferators.
[0016] Widespread expression of PPAR.delta. is observed in both the
embryo and in adult tissues. This observation suggests that this
isoform may play a general "housekeeping" role. In contrast,
PPAR.gamma. is observed to be expressed almost exclusively in the
adrenal and spleen. The expression of all three PPAR isoforms in
the adrenal is particularly intriguing, since diseases which result
in peroxisome dysfunction (e.g. adrenoleukodystrophy and Zellweger
syndrome) cause gross morphological changes in adrenal cells and,
eventually, adrenal deficiency. These observations suggest a
critical role for peroxisomes in this tissue (Vamecq and Draye,
supra). Interestingly, peroxisomes can be induced to proliferate in
hamster adrenals in response to treatment with adrenocorticotropic
hormone and corticosteroids (Black and Russo, Amer. J. Anatomy
159:85-120 (1980)), indicating the presence of adrenal-specific
signaling pathway(s) for peroxisome proliferation. The differential
expression of PPAR.gamma. in the adrenal suggests that this isoform
may respond to an adrenal-enriched ligand.
[0017] Accordingly, in accordance with the present invention, there
are provided isolated mammalian peroxisome proliferator-activated
receptor subunit proteins of the .alpha. or .delta. subtype and
functional fragments thereof.
[0018] As employed herein, the phrase "mammalian peroxisome
proliferator-activated receptor subunit proteins of the .gamma. or
.delta. subtype" refers to isolated and substantially purified as
well as recombinantly produced proteins which are members of the
steroid/thyroid superfamily of receptors, and which mediate the
pleiotropic effects of peroxisome proliferators (such as medium and
bong-chain fatty acids). Such receptors participate in the
formation of heterodimeric species with retinoid X receptors (RXRs)
and comprise an amino-terminal domain, a DNA binding domain, and a
ligand binding domain. Also contemplated within this definition are
variants thereof encoded by mRNA generated by alternative splicing
of a primary transcript.
[0019] Use of the terms "recombinantly produced", "isolated" or
"substantially pure" in the present specification and claims as a
modifier of DNA, RNA, polypeptides or proteins means that the
modified substances have been produced by the hand of man, and thus
are separated from their native in vivo cellular environment. As a
result of this human intervention, the
recombinant/isolated/substantially pure DNAs, RNAs, polypeptides
and proteins of the invention are useful in ways that the naturally
occurring DNAs, RNAs, polypeptides or proteins are not, for
example, in assays to identify selective drugs or compounds.
[0020] The novel receptors of the present invention also can be
included as part of a panel of receptors which are screened to
determine the selectivity of interaction of proposed agonists or
antagonists of other steroid hormone receptors. Thus, a compound
which is believed to interact selectively, for example, with the
glucocorticoid receptor, should not have any substantial effect on
any other receptors, including invention receptors. However, if
such a proposed compound does interact with the invention
receptors, then the probability of side effects caused by the
activation of other receptors in addition to the target receptor,
is clearly indicated. For example, the use of many drugs in the
treatment of hormone-related disorders is currently restricted by
side effects caused by the activation of "non-target" receptors.
Employment of the invention receptors in a panel of receptors in a
screen to determine the selectivity of interaction of potential
bigands provides a means to identify receptor-specific ligands that
are therapeutically superior than currently used ligands that cause
unwanted side effects.
[0021] As used herein, the term splice variant refers to variant
PPAR encoding nucleic acid(s) produced by differential processing
of primary transcript(s) of genomic DNA, resulting in the
production of more than one mRNA. cDNA derived from differentially
processed primary transcript will encode PPAR receptor proteins
that have regions of complete amino acid identity and regions
having different amino acid sequences. Thus, the same genomic
sequence can bead to the production of multiple, related mRNAs and
corresponding proteins. Both the resulting mRNAs and proteins are
referred to herein as "splice variants".
[0022] Accordingly, also contemplated within the scope of the
present invention are nucleic acids that encode mammalian PPAR
receptor subunit proteins as defined above, but that by virtue a
degenerate genetic code do not necessarily hybridize to the nucleic
acids set forth in SEQ ID NOs: 1 or 3 under specific hybridization
conditions. Nucleic acid fragments encoding invention receptor
subunit proteins are capable of forming a functional heterodimer
with one or more RXR receptor protein isoform(s). Typically, unless
a PPAR receptor protein is encoded by mRNA that arises from
alternative splicing (i.e., a splice variant), PPAR receptor
encoding DNA and encoded protein share substantial sequence
homology with at least one of the PPAR receptor-encoding DNAs and
encoded proteins described herein. It is understood that DNA or RNA
encoding a splice variant may share less than 90% overall sequence
homology with the DNA or RNA provided herein, but include regions
of nearly 100% homology to a DNA fragment described herein, and
encode an open reading frame that includes start and stop codons
and encodes a functional PPAR receptor protein.
[0023] Exemplary nucleic acid sequences encoding mammalian
peroxisome proliferator-activated receptor subunit proteins of the
.gamma. subtype are represented by nucleotide sequences which
encode substantially the same amino acid sequence as set forth in
SEQ ID NO:2. Presently preferred sequences encode the same amino
acid sequence as set forth in SEQ ID NO:2.
[0024] Exemplary nucleic acid sequences can alternatively be
characterized as those nucleotide sequences which encode mammalian
peroxisome proliferators-activated receptor subunit proteins of the
.gamma. subtype and hybridize under high stringency conditions to
SEQ ID NO: 1.
[0025] Exemplary nucleic acid sequences encoding mammalian
peroxisome proliferator-activated receptor subunit proteins of the
.delta. subtype are represented by nucleotides which encode
substantially the same amino acid sequence as set forth in SEQ ID
NO:4. Presently preferred sequences encode the same amino acid
sequence as set forth in SEQ ID NO:4.
[0026] Especially preferred sequences are those which have
substantially the same nucleotide sequence as that set forth in SEQ
ID NO:1.
[0027] Exemplary nucleic acid sequences can alternatively be
characterized as those nucleotide sequences which encode mammalian
peroxisome proliferators-activated receptor subunit proteins of the
.delta. subtype and hybridize under high stringency conditions to
SEQ ID NO:3.
[0028] Especially preferred nucleic acid sequences are those which
have substantially the same nucleotide sequence as the coding
sequences in SEQ ID NO:3.
[0029] The phrase "stringency of hybridization" is used herein to
refer to conditions under which polynucleic acid hybrids are
stable. As known to those of skill in the art, the stability is
reflected in the melting temperature (T.sub.m) of the hybrids.
T.sub.m can be approximated by the formula: 81.5.degree.
C.-16.6(log.sub.10[Na.sup.+])+0.41(%G+C)-600/1, where 1 is the
length of the hybrid in number of nucleotides. T.sub.m decreases
approximately 1-1.5.degree. C. with every 1% decrease in sequence
homology. In general, the stability of a hybrid is a function of
sodium ion concentration and temperature. Typically, the
hybridization reaction is initially performed under conditions of
low stringency, followed by washes of varying, but higher,
stringency. Reference to hybridization stringency relates to such
washing conditions. Thus, as used herein: [0030] (1) HIGH
STRINGENCY refers to conditions that permit hybridization of only
those nucleic acid sequences that form stable hybrids in 0.01 8M
NaCl at 65.degree. C. (i.e., if a hybrid is not stable in 0.01 8M
NaCl at 65.degree. C., it will not be stable under high stringency
conditions, as contemplated herein). High stringency conditions can
be provided, for example, by hybridization in 50% formamide,
5.times. Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree.
C., followed by washing in 0.1.times.SSPE, and 0.1% SDS at
65.degree. C.; [0031] (2) MODERATE STRINGENCY refers to conditions
that permit hybridization in 50% formamide, sx Denhart's solution,
5.times.SSPE, 0.2% SDS at 42.degree. C., followed by washing in
0.2.times.SSPE, 0.2% SDS, at 65.degree. C.; and [0032] (3) LOW
STRINGENCY refers to conditions that permit hybridization in 10%
formamide, 5.times. Denhart's solution, 6.times.SSPE, 0.2% SDS at
42.degree. C., followed by washing in 1.times.SSPE, 0.2% SDS, at
50.degree. C. It is understood that these conditions may be varied
using a variety of buffers and temperatures well known to skilled
artisans.
[0033] As used herein, the phrase "substantial sequence homology"
refers to nucleotide sequences which share at least about 90%
identity, and amino acid sequences which typically share more than
95% amino acid identity. It is recognized, however, that proteins
(and DNA or mRNA encoding such proteins) containing less than the
above-described level of homology arising as splice variants or
that are modified by conservative amino acid substitutions (or
substitution of degenerate codons) are contemplated to be within
the scope of the present invention.
[0034] As used herein, the phrase "substantially the same" refers
to nucleotide sequences, ribonucleotide sequences, or amino acid
sequences, that have slight and non-consequential sequence
variations from the actual sequences disclosed herein. Species that
are "substantially the same" are considered to be equivalent to the
disclosed sequences, and as such are within the scope of the
appended claims. In this regard, "slight and non-consequential
sequence variations" mean that sequences that are substantially the
same as invention sequences disclosed and claimed herein, are
functionally equivalent to the sequences disclosed and claimed
herein. Functionally equivalent sequences will function in
substantially the same manner to produce substantially the same
results as the nucleic acid and amino acid sequences disclosed and
claimed herein. Specifically, functionally equivalent nucleic acids
encode proteins that have conservative amino acid variations, such
as substitution of a non-polar residue for another non-polar
residue or a charged residue for a similarly charged residue. These
changes are recognized by those of skill in the art as
modifications that do not substantially alter the tertiary
structure of the protein.
[0035] Fragments of invention nucleic acid sequences are useful as
hybridization probes, wherein such fragments comprise at least 14
contiguous nucleotides of the above-described nucleic acids, and
wherein the fragment is labeled with a detectable substituent.
Suitable detectable substituents can be readily determined by those
of skill in the art, and include such species as radiolabeled
molecules, fluorescent molecules, enzymes, ligands, and the
like.
[0036] As used herein, a probe is single- or doublestranded DNA or
RNA that has a sequence of nucleotides that includes at least 14
contiguous bases that are the same as (or the complement of) any 14
or more contiguous bases set forth in SEQ ID NOs:1 or 3. Preferred
regions for the construction of probes include those regions
predicted to encode a DNA binding domain. Such regions are
preferred because they are most highly conserved among members of
the steroid/thyroid superfamily of receptors.
[0037] As a particular application of the invention sequences,
genetic screening can be carried out using the nucleic acid
sequences of the invention as probes. Thus, nucleic acid samples
from patients having conditions suspected of involving
alteration/modification of any one or more of the PPAR receptor
subtypes can be screened with appropriate probes to determine if
abnormalities exist with respect to the endogenous PPAR receptor
proteins.
[0038] In accordance with yet another embodiment of the present
invention, there are provided vectors comprising nucleic acid
sequences, as well as cells and vectors containing such sequences.
Such host cells, including bacterial, yeast and mammalian cells can
be used for expressing invention nucleic acids to produce PPAR
receptor protein(s). Incorporation of cloned DNA into a suitable
expression vector, transfection of eukaryotic cells with a plasmid
vector or a combination of plasmid vectors, each encoding one or
more distinct genes, and selection of transfected cells are well
known in the art (see, e.g., Sambrook et al. (1989) Molecular
Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press). Heterologous DNA may be introduced into host
cells by any method known to those of skill in the art, such as
transfection by CaPO.sub.4 precipitation with a vector encoding the
heterologous DNA (see, e.g., Wigler et al. (1979) Proc. Natl. Acad.
Sci. 76:1373-1376), DEAE-dextran, electroporation, microinjection,
or lipofectamine (GIBCO BRL #18324-012). Transfected host cells can
then be cultured under conditions whereby the receptor subunit
protein(s) encoded by the DNA is (are) recombinantly expressed.
[0039] The present invention further provides a mammalian
peroxisome proliferator-activated receptor, expressed recombinantly
in a host cell. The receptor comprises at least one PPAR subunit,
wherein the PPAR subunit is PPAR.gamma. or PPAR.delta., and at
least one retinoid X receptor isoform. The invention receptor has
the ability to repress PPAR.alpha.-mediated responses activated by
Wy 14,643.
[0040] Also provided by the present invention are mammalian
peroxisome proliferator-activated subunit proteins, expressed
recombinantly in a host cell wherein the receptor subunits have
substantially the same amino acid sequence as set forth in SEQ ID
NOs: 2 or 4.
[0041] In accordance with still another embodiment of the present
invention, there is provided a method for the recombinant
production of mammalian peroxisome proliferator-activated receptor
proteins comprising at least one PPAR subunit of the .gamma. or
.delta. subtype, or functional fragments thereof. Such method
comprises expressing the above-described nucleic acid(s) in a
suitable host cell.
[0042] In accordance with still another embodiment of the present
invention, there is provided a method to identify clones encoding
mammalian peroxisome proliferatoractivated receptor subunit
proteins of the .gamma. or .delta. subtype, or functional fragments
thereof. Such method comprises screening a genomic or cDNA library
with an invention nucleic acid probe under low stringency
hybridization conditions, and identifying those clones which
display a substantial degree of hybridization to said fragment.
[0043] Nucleic acids encoding mammalian peroxisome
proliferator-activated receptor subunit protein of the .gamma. or
.delta. subtype, or functional fragments thereof may be isolated by
screening suitable human cDNA or human genomic libraries under
suitable hybridization conditions with nucleic acids disclosed
herein (including nucleotide sequences derived from SEQ ID NOs:1 or
3). Suitable libraries can be prepared from appropriate tissue
samples, e.g., brain tissue, heart tissue, intestinal tissue,
kidney tissue, liver tissue, spleen tissue, and the like. The
library can be screened with nucleic acid including substantially
the entire receptor-encoding sequence thereof, or the library may
be screened with a suitable probe, as described above.
[0044] After screening the library, positive clones are identified
by means of a hybridization signal; the identified clones are
characterized by restriction enzyme mapping and/or DNA sequence
analysis, and then examined, by comparison with the sequences set
forth herein to ascertain whether they encode a complete PPAR
receptor subunit protein (i.e., if they include translation
initiation and termination codons). If the selected clones are
incomplete, they may be used to rescreen the same or a different
library to obtain overlapping clones. If the library is genomic,
then the overlapping clones may include exons and introns. If the
library is a cDNA library, then the overlapping clones will include
an open reading frame. In both instances, complete clones may be
identified by comparison with the DNA and encoded proteins provided
herein.
[0045] The ligand-binding domain (LBD) of nuclear hormone receptors
is a complex multifunctional unit containing subdomains for
dimerization, transcriptional suppression and hormone-induced
transactivation (Forman and Samuels, Mol. Endocrinol.
4:1293-1301(1990)). The dimerization domain includes a series of
heptad repeats flanked by sequences required for ligand binding.
Thus, the dimerization domain is embedded within the larger LBD.
This structural arrangement raises the possibility that
dimerization may serve as an allosteric modulator of ligand binding
and transactivation. It has previously been shown that the
Drosophila ecdysone receptor (EcR) acquires ligand binding activity
after heterodimerization with USP (Drosophila homolog of RXR; see
Yao et al., in Nature 366:476-479 (1993)). Thus, differential
interactions among receptor LBDs can either restrict, redirect or
lead to an acquisition of new ligand binding phenotypes.
[0046] It has recently been shown that PPAR.alpha. binds to its
cognate response elements as a heterodimer with the RXR (see
Kliewer et al., supra, Gearing et al., supra, or Keller et al.,
supra). The resulting PPAR.alpha.-RXR complex can respond to both
peroxisome proliferators and 9-cis retinoic acid (see Kliewer et
al., (1992), supra). It has now been found that PPAR.gamma. and
PPAR.delta. also cooperate with RXR in the formation of
heterodimers, and in binding to DNA as heterodimers. Ultimately,
the regulation of peroxisome physiology is likely a consequence of
a complex interplay among the multiple PPAR and RXR isoforms and
the ligands for these receptors.
[0047] In accordance with the present invention, there are provided
combinations of receptors comprising at least two different members
of the steroid/thyroid superfamily of receptors, wherein one
receptor is either PPAR.gamma. or PPAR.delta., and wherein said
receptors are associated in the form of a multimer, preferably a
heterodimer. A particularly preferred combination of receptors is a
heterodimer comprising either PPAR.gamma. or PPAR.delta. and a
subtype of RXR.
[0048] Combinations contemplated by the present invention can
broadly be referred to as "multimeric species," which is intended
to embrace all of the various oligomeric forms in which members of
the steroid/thyroid superfamily of receptors (including fragments
thereof comprising the dimerization domains thereof) are capable of
associating in combination with either PPAR.gamma. or PPAR.delta..
Thus, reference to "combinations" of steroid receptors or
"multimeric" forms of steroid receptors includes homodimeric
combinations of a single PPAR.gamma. or PPAR.delta. receptor
(including fragments thereof comprising the dimerization domains
thereof), heterodimeric combinations of either a PPAR.gamma. or
PPAR.delta. receptor and another different receptor (including
fragments thereof comprising the dimerization domains thereof),
homotrimeric combinations of a single PPAR.gamma. or PPAR.delta.
receptor (including fragments thereof comprising the dimerization
domains thereof), heterotrimeric combinations of two or three
different receptors including PPAR.gamma. or PPAR.delta. (including
fragments thereof comprising the dimerization domains thereof)
homotetrameric combinations of a single PPAR.gamma. or PPAR.delta.
receptor (including fragments thereof comprising the dimerization
domains thereof), heterotetrameric combinations of two or more
different receptors including PPAR.gamma. or PPAR.delta. (including
fragments thereof comprising the dimerization domains thereof), and
the like.
[0049] As employed herein, the phrase "members of the
steroid/thyroid superfamily of receptors" (also known as "nuclear
receptors" or "intracellular receptors") refers to hormone binding
proteins that operate as ligand-dependent transcription factors,
including identified members of the steroid/thyroid superfamily of
receptors for which specific ligands have not yet been identified
(referred to hereinafter as "orphan receptors"). These hormone
binding proteins have the intrinsic ability to bind to specific DNA
sequences. Following binding, the transcriptional activity of
target gene (i.e., a gene associated with the specific DNA
sequence) is modulated as a function of the ligand bound to the
receptor.
[0050] The DNA-binding domains of all of these nuclear receptors
are related, consisting of 66-68 amino acid residues, and
possessing about 20 invariant amino acid residues, including nine
cysteines. A member of the superfamily can be identified as a
protein which contains the above-mentioned invariant amino acid
residues, which are part of the DNA-binding domain of such known
steroid receptors as the human glucocorticoid receptor (amino acids
421-486), the estrogen receptor (amino acids 185-250), the
mineralocorticoid receptor (amino acids 603-668), the human
retinoic acid receptor (amino acids 88-153). The highly conserved
amino acids of the DNA-binding domain of members of the superfamily
are well-known as set forth, for example in PCT WO 94/01558. Thus,
the DNA-binding domain is a minimum of 66 amino acids in length,
but can contain several additional residues.
[0051] Exemplary members of the steroid/thyroid superfamily of
receptors contemplated for use in the practice of the present
invention (including the various isoforms thereof) include steroid
receptors such as mineralocorticoid receptor, progesterone
receptor, androgen receptor, vitamin D.sub.3 receptor, and the
like; plus retinoid receptors, such as the various isoforms of RAR
(e.g., RAR.alpha., RAR.beta., or RAR.gamma.), the various isoforms
of RXR (e.g., RXR.alpha., RXR.beta., or RXR.gamma.), and the like;
thyroid receptors, such as TR.alpha., TR.beta., and the like; as
well as other gene products which, by their structure and
properties, are considered to be members of the superfamily, as
defined hereinabove, including the various isoforms thereof.
Examples of orphan receptors include HNF4 [see, for example, Sladek
et al., in Genes & Development 4: 2353-2365 (1990)], the COUP
family of receptors [see, for example, Miyajima et al., in Nucleic
Acids Research 16: 11057-11074 (1988), and Wang et al., in Nature
340: 163-166 (1989)], COUP-like receptors and COUP homologs, such
as those described by Mlodzik et al., in Cell 60: 211-224 (1990)
and Ladias et al., in Science 251:561 -565 (1991), the
ultraspiracle receptor example, [see, for example, Oro et al., in
Nature 347: 298-301 (1990)], and the like. Presently preferred
members of the superfamily for use in the practice of the present
invention are the various isoforms of RXR (e.g., RXR.alpha.,
RXR.beta., or RXR.gamma.).
[0052] The formation of multimeric (e.g., heterodimeric) species
can modulate the ability of the first receptor to trans-activate
transcription of genes maintained under expression control in the
presence of ligand for said first receptor. The actual effect on
activation of transcription (i.e., enhancement or repression of
transcription activity) will vary depending on the receptor species
which is combined with either a PPAR.gamma. or PPAR.delta. receptor
to form the multimeric species, as well as on the response element
with which the multimeric species interacts.
[0053] In accordance with the present invention, there are provided
multimeric receptor species which belong to the steroid/thyroid
superfamily of receptors, comprising at least the dimerization
domain of at two different members of the steroid/thyroid
superfamily of receptors, wherein one of the members is selected
from the invention PPAR.gamma. or PPAR.delta..
[0054] As employed herein, the tern "dimerization domain" of a
member of the steroid/thyroid superfamily of receptors refers to
that portion of the receptor which is believed to be involved in
the formation of multimeric receptor species. This domain typically
comprises the carboxy-terminal portion of the receptor, i.e., that
portion of a receptor which is 3' with respect to the DNA-binding
domain of the receptor. See, e.g., Evans, in Science 240:889-895
(1988), and Forman and Samuels, Mol. Endocrinol. 4:1293-1301
(1990). Presently preferred members of the superfamily for use in
deriving the dimerization domain are the various isoforms of RXR
(e.g., RXR.alpha., RXR.beta., or RXR.gamma.).
[0055] In accordance with the present invention, there are also
provided heterodimer complexes comprising either PPAR.gamma. or
PPAR.delta. and a silent partner therefor.
[0056] As employed herein, the term "silent partner" refers to
members of the steroid/thyroid superfamily of receptors which are
capable of forming heterodimeric species with either PPAR.gamma. or
PPAR.delta., wherein the silent partner of the heterodimer does not
have any ligand bound to the ligand-binding domain (LBD) when the
silent partner is complexed with a PPAR subtype (i.e., only the
PPAR copartner of the heterodimer binds ligand). Presently
preferred silent partners for use in the practice of the present
invention are the various isoforms of RXR (e.g., RXR.alpha.,
RXR.beta., or RXR.gamma.).
[0057] In accordance with a further embodiment of the present
invention, there is provided a method for screening compounds to
determine those which bind to mammalian peroxisome
proliferator-activated receptor proteins comprising at least one
PPAR subunit of the .gamma. or .delta. subtype, or functional
fragments thereof. Such method comprises employing receptor
protein(s) of the invention in a binding assay, which comprises,
contacting receptor protein(s) of the invention with test compound,
and identifying those compounds which bind to invention receptor
protein(s).
[0058] In accordance with a still further embodiment of the present
invention, there is provided a bioassay for evaluating whether test
compounds are agonists for receptor proteins of the invention, or
functional modified forms of said receptor protein(s). Such
bioassay comprises: [0059] (1) contacting suitable host cells
expressing said receptor protein with test compound under
physiological conditions, wherein said host cells contain DNA
encoding a reporter protein, wherein said DNA is operatively linked
to a PPAR-response element; [0060] (2) monitoring said host cells
for expression of reporter gene, wherein expression of reporter
protein reflects transcriptional activity of the receptor protein
and, therefore, the presence of an activated receptor-ligand
complex.
[0061] In accordance with yet another embodiment of the present
invention, there is provided a bioassay for evaluating whether test
compounds are antagonists for receptor proteins of the invention,
or functional modified forms of said receptor protein(s). Such
bioassay comprises:
[0062] contacting suitable host cells with [0063] (i) increasing
concentrations of at least one compound whose ability to inhibit
the transcription activation activity of agonists of mammalian
peroxisome proliferator-activated receptor proteins of the .gamma.
or .delta. subtype is sought to be determined, and [0064] (ii) a
fixed concentration of at least one agonist for said receptor
protein(s) or functional modified forms thereof, [0065] wherein
suitable test cells express mammalian peroxisome
proliferator-activated receptor proteins comprising at least one
PPAR subunit of the .gamma. or .delta. subtype and DNA encoding a
reporter protein, wherein said DNA is operatively linked to a
PPAR-response element; and thereafter
[0066] assaying for evidence of transcription of said reporter gene
in said cells as a function of the concentration of said compound
in said culture medium, thereby indicating the ability of said
compound to inhibit activation of transcription by agonists of
mammalian peroxisome proliferator-activated receptor proteins
comprising at least one PPAR subunit of the .gamma. or .delta.
subtype.
[0067] In accordance with a still further embodiment of the present
invention, there is provided a method for identifying ligands
selective for heterodimers comprising either PPAR.gamma. or
PPAR.delta. and a silent partner therefor. Such method
comprises
[0068] comparing the level of expression of reporter when cells
containing a reporter construct, either PPAR.gamma. or PPAR.delta.
and silent partner therefor are exposed to test compound, relative
to the level of expression of reporter when cells containing a
reporter construct, either PPAR.gamma. or PPAR.delta. and a member
of the steroid/thyroid superfamily which is not a silent partner
therefor are exposed to test compound, and
[0069] selecting those compounds which activate only the
combination of either PPAR.gamma. or PPAR.delta. and silent partner
therefor.
[0070] In accordance with yet another embodiment of the present
invention, there are provided antibodies generated against the
invention proteins. Such antibodies can be employed for studying
receptor tissue localization, subunit composition, structure of
functional domains, as well as in diagnostic applications,
therapeutic applications, and the like. Preferably, for therapeutic
applications, the antibodies employed will be monoclonal
antibodies.
[0071] The above-described antibodies can be prepared employing
standard techniques, as are well known to those of skill in the
art, using the invention receptor proteins or portions thereof as
antigens for antibody production. Both anti-peptide and anti-fusion
protein antibodies can be used Trends Pharmacol Sci. vol.
12:338-343; Current Protocols in Molecular Biology (Ausubel et al.,
eds.) John Wiley and Sons, New York (1989)]. Factors to consider in
selecting portions of the invention receptor protein subunit
sequences for use as immunogen (as, for example, a synthetic
peptide or a recombinantly produced bacterial fusion protein)
include antigenicity, accessibility (i.e., internal or external
domains), uniqueness to the particular protein subunit, and the
like.
[0072] The availability of sequence-specific antibodies enables use
of immunohistochemical techniques to monitor the distribution and
expression density of various protein subunits (e.g., in normal
versus diseased brain tissue). Such antibodies can also be employed
for diagnostic and therapeutic applications.
[0073] In accordance with yet another embodiment of the present
invention, there are provided methods for modulating processes
mediated by mammalian peroxisome proliferator-activated receptor
proteins comprising at least one PPAR subunit of the .gamma. or
.delta. subtype. Such methods comprise contacting mammalian
peroxisome proliferatoractivated receptor proteins of the .gamma.
or .delta. subtype with an effective, modulating amount of agonist,
antagonist or antibody according to the present invention.
[0074] The antibodies, agonists and/or antagonists of the invention
can be administered to a subject employing standard methods, such
as, for example, by intraperitoneal, intramuscular, intravenous, or
subcutaneous injection, implant or transdermal modes of
administration, and the like. One of skill in the art can readily
determine dose forms, treatment regiments, etc, depending on the
mode of administration employed.
[0075] Processes which are mediated by mammalian peroxisome
proliferator-activated receptor proteins of the .gamma. or .delta.
subtype include, for example, macrophage production in the spleen
which is believed to be important in atherosclerosis.
[0076] The invention will now be described in greater detail with
reference to the following non-limiting examples.
EXAMPLE 1
Screening of cDNA Libraries
[0077] PPAR.gamma. was isolated by screening an adult mouse liver
.lamda.ZAP cDNA library (Stratagene) with a synthetic
oligonucleotide (GGNTTYCAYTAYGGNGTNCAYCG; SEQ ID NO: 5) under
conditions previously described by Blumberg et al., in Proc. Natl.
Acad. Sci. USA 89:2321-2325 (1992). This oligonucleotide is a
mixture of all possible DNA sequences encoding the amino acid
sequence GFHYGVHA (SEQ ID NO:6), a sequence present in the loop of
the first zinc finger in the Xenopus PPAR.alpha. PPAR.beta. and
PPAR.gamma. isoforms.
[0078] PPAR.delta. was isolated by screening an E6.5 mouse
.lamda.ZAPII cDNA library (a gift of D. E. Weng and J. D. Gerhart,
Johns Hopkins University) under low stringency conditions with a
cDNA fragment encoding the human retinoic acid receptor aDNA
binding domain (Mangelsdorf et al., Nature 345:224-229 (1990)). In
both screens, positive clones were converted to plasmids by the
automatic excision process.
EXAMPLE 2
Cotransfection Assay
[0079] The mammalian expression vectors pCMX-PPAR.alpha.,
pCMX-PPAR.gamma. and pCMX-PPAR.delta. were constructed by inserting
the cDNA inserts of PPAR.alpha., PPAR.gamma., and PPAR.delta. into
pCMX as previously described by Umesono et al., in Cell
65:1255-1266 (1991)). Construction of the reporter
PPRE.sub.3-TK-LUC has also been previously described by Kliewer et
al., (1992) supra. Cotransfection assays in CV-1 cells were done in
48 well plates using N-[1-(2,3-dioleoyloxy)-propyl [N,N,N-trimethyl
ammonium methyl sulfate (DOTAP) according to the manufacturer's
instructions (Boehringer Mannheim). Transfections contained 10 ng
of receptor expression plasmid vector, 20 ng of the reporter
PPRE.sub.3-TK-LUC, 60 ng of pCMX-.beta.GAL (.beta.-galactosidase)
as an internal control, and 210 ng of carrier plasmid pGEM. Cells
were incubated in the presence of DOTAP for 8 hours, washed, and
incubated in the presence of peroxisome proliferators or fatty
acids for 36 hours. Cell extracts were prepared and assayed for
luciferase and .beta.-galactosidase activity as previously
described (Umesono, supra). All experimental points were done in
triplicate.
EXAMPLE 3
Northern Analysis
[0080] Preparation of poly(A).sup.+RNA from rat tissues and
Northern analysis were performed as previously described
(Mangelsdorf et al., supra). Thus, Northern blot analysis of PPAR
mRNA was carried out employing adult and embryonic tissue. Adult
male rat tissues and mouse embryos from gestation day 10.5 to 18.5
were employed. The exposure time for each of the blots was 48
hours. The sizes of the transcripts, based on RNA size markers,
were 8.5 kb (PPAR.alpha.), 1.9 kb (PPAR.gamma.), and 3.5 kb
(PPAR.delta.).
EXAMPLE 4
DNA Binding Assays
[0081] Gel mobility shift assays were performed as previously
described by Kliewer et al. (1992) supra. PPAR.alpha., PPAR.gamma.,
PPAR.delta., RXR.beta., RXR,.delta. and RXR.gamma. were synthesized
in vitro using the TNT coupled transcription/translation system
(Promega) according to the manufacturer's instructions.
EXAMPLE 5
Isolation of Three Murine PPAR Isoforms
[0082] The function of peroxisome proliferators has been most
extensively studied in rodents, where treatment with these
compounds results in marked increases in peroxisome size and number
and concomitant increases in the expression of the genes encoding
the enzymes of the peroxisomal .beta.-oxidation pathway. To gain
insight into the function of PPAR isoforms, mouse embryonic and
adult liver libraries were screened for PPAR.alpha.-related gene
products. In addition to PPAR.alpha., two types of
PPAR.alpha.-related clones were isolated.
[0083] The first clone encodes a 475-amino acid protein that is 56%
identical to mouse (m)PPAR.alpha. and 76% identical to Xenopus
(x)PPAR.gamma.. Since this clone is 97% and 84% identical to the
DNA binding and ligand binding domains of xPPAR.gamma.,
respectively, it is designated mPPAR.gamma. (see SEQ ID NOs:1 and
2).
[0084] The second clone encodes a 440-amino acid protein that is
closely related to NUC-1 (see SEQ ID NOs:3 and 4, and FIG. 1), a
PPAR.alpha.-related receptor recently isolated from a human
osteosarcoma library (see Schmidt et al., in Mol. Endo. 6:1634-1641
(1992)). Since this second clone is not highly homologous to any of
the previously identified PPAR isoforms (i.e., mPPAR.alpha.,
xPPAR.alpha., xPPAR.beta. or xPPAR.gamma., see FIG. 1), it appears
to represent a novel receptor, and is, therefore, designated
mPPAR.delta.. Of the approximately 50 positives characterized
during the course of screening, no mouse homolog of xPPAR.beta. was
identified.
EXAMPLE 6
PPAR.alpha., PPAR.gamma., and PPAR.delta. are Differentially
Expressed in the Adult and Embryo
[0085] The expression patterns of the murine PPAR isoforms were
examined in the embryo and adult. Northern analysis of
poly(A).sup.+ RNA isolated from adult male rat tissues revealed
differential yet overlapping patterns of expression of the three
isoforms. Both PPAR.alpha. and PPAR.delta. are widely expressed,
with PPAR.alpha. message levels highest in the liver, kidney,
heart, and adrenal, and PPAR.delta. message highest in the heart,
adrenal, and intestine. In contrast, PPAR.gamma. displays a more
restricted distribution pattern, with abundant expression in only
the adrenal and spleen, although message is also detectable in the
heart, kidney, and intestine.
[0086] The developmental expression of the PPAR isoforms was also
examined through Northern analysis of whole mouse embryo RNA.
PPAR.alpha. and PPAR.gamma. displayed similar expression patterns
during mouse embryogenesis, with message first appearing at day
13.5 postconception and increasing until birth. In contrast,
PPAR.delta. message was abundant at all the embryonic time points
tested, suggesting a broad role for this isoform during
development. Thus, the PPAR isoforms are seen to be differentially
expressed in both the embryo and the adult.
EXAMPLE 7
Evidence for Pharmacological Differences Between PPAR.alpha.,
PPAR.gamma. and PPAR.beta.
[0087] The relatively high degree of conservation within the ligand
binding domains of PPAR.alpha., PPAR.gamma. and PPAR.delta.
suggested that these PPAR isoforms might respond to the same
activators. Accordingly, each of the PPAR isoforms was first tested
for responsiveness to Wy 14,643, a peroxisome proliferator and
potent activator of PPAR.alpha. (Reddy and Laiwani, Crit. Rev.
Toxicol. 12:1-58 (1983)). Cotransfection of PPAR.alpha. expression
plasmid resulted in a dramatic (>100-fold) increase in
activation of a reporter construct containing three copies of the
acyl-CoA oxidase PPRE (AOX-PPRE) upstream of the thymidine kinase
promoter driving luciferase expression (PPRE.sub.3-TK-LUC) in
response to Wy 14,643 (FIG. 2).
[0088] In contrast, no activation of reporter expression was seen
in the presence of Wy 14,643 upon cotransfection of PPAR.gamma. or
PPAR.delta. expression plasmids (FIG. 2). This lack of activation
is unlikely to reflect differences in binding site specificity, as
each of the PPAR isoforms bound efficiently to the AOX-PPRE as a
heterodimer with RXR (as determined by gel mobility shift assays
done using in vitro synthesized PPAR.alpha., PPAR.gamma., and
PPAR.delta., and/or RXR.gamma., and .sup.32P-labeled AOX-PPRE
oligonucleotide). Additional experiments revealed that
overexpression of PPAR.gamma. and PPAR.delta. interfered with the
ability of PPAR.alpha. to activate through the AOX-PPRE (FIG. 3).
Thus, both PPAR.gamma. and PPAR.delta. are expressed and can
function as dominant repressors of PPAR.alpha.-mediated
responsiveness to Wy 14,643.
[0089] Since no activation of PPAR.gamma. and PPAR.delta. was
detected with Wy 14,643, other potential activators were tested,
including a broad spectrum of peroxisome proliferators and fatty
acids. As shown in FIG. 4, significant activation of PPAR.gamma.
was obtained upon treatment with LY-171883, a leukotriene
antagonist and peroxisome proliferator which lacks the carboxyl
group typically found in this class of compounds (Foxworthy and
Eacho, Biochem. Pharmacology 42:1487-1491 (1991)). Conversely, no
activation of PPAR.gamma. was seen in the presence of linoleic acid
(FIG. 4).
[0090] In contrast to the results obtained with PPAR.gamma.,
PPAR.delta. was activated in the presence of linoleic acid, but was
not activated upon treatment with LY-171883. Both LY-171883 and
linoleic acid are strong activators of PPAR.alpha. (FIG. 4).
Interestingly, each of the three PPAR isoforms was activated with a
distinct rank order of efficacy by these compounds:
[0091] PPAR.alpha.. [0092] Wy 14,643>L,Y-171883>linoleic
acid;
[0093] PPAR.gamma.. [0094] LY 171883>linoleic acid>Wy
14,643;
[0095] PPAR.delta.: [0096] linoleic acid>LY-171883 and Wy
14,643. See FIG. 4. These data provide evidence that PPAR.gamma.
and PPAR.delta. can function as regulated activators of gene
expression and that the three PPAR isoforms are pharmacologically
distinct.
[0097] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
Sequence CWU 1
1
* * * * *